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Órgano oficial de difusión de la Federación Mexicana de Patología Clínica, AC y de la Asociación Latinoamericana de Patología Clínica/Medicina de Laboratorio

2013, Number 2

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Rev Mex Patol Clin Med Lab 2013; 60 (2)

A rapid, sensitive, fast and easy to interpret antivirogram by a bioluminiscent assay for HIV-1

Lara HH, Ixtepan-Turrent L, Rivera-Silva G, González-Salazar F, Moreno-Treviño G

Language: Spanish
References: 37
Page: 79-90
PDF size: 103.72 Kb.


ABSTRACT

Objective: To demonstrate that our assay is sensitive capable to detect 10 HIV infected cells per well and that is feasible to quantify HIV-1 resistance in seropositive patients.
Method: To compare 3 different phenotypic assays by quantifying their sensitivities, first we co-cultivated HeLa-CD4-LTR/β-gal cells with infected PBMCs at different concentrations diluted 1:2 from 4,000 cells per well in order to elucidate the minimum quantity of cells needed for the assay to be detected as positive. On day one in a 96 well plate we seeded HeLa-CD4-LTR/β-gal (5 x 104) cells in 50 HeLa-CD4-LTR/β-gal (5 x 104) of DMEM. 24 h later we added the following antiretrovirals Abacavir, Didanosina, Lamivudina, Tenofovir, Efavirenz, Nevirapina, Enfurtivide, Amprenavir, Atazanavir, Indinavir, Lopinavir, Nelfinavir, Ritonavir, y Saquinavir. To obtain dose response we diluted 1:2 the drugs. Then we added the four patients’ PBMCs infected with HIV-1, then it is incubated at 37 oC for 24 h then the plates are readed after the addition of luciferasa to the 96 well plate at day 3.
Results: HIV infection was quantified as very sensitive, rapid and specific. We obtained the IC50 for HIVwt OR wild type and VIHRV or resistant virus. Our method was more sensitive than the MT-2 assay (by 200 fold) and more sensitive than Blue cells (by 30 fold). The assay quantified the infection by PBMC+ cells from seropositive patients being able to classify the sensitivities and HIV resistance. In four patients was detected resistance to protease inhibitors, light resistance to NRTI, moderate resistance to NNRTI, sensitive to Enfurtivide and two patients sensitive to Lamivudine.
Conclusions: In Mexico there is no antivirogram for HIV-1. The assay that we tested is a Bioluminiscent assay that detect resistant virus and is capable to quantify the IC50 of HIV-1 with precision. It is a valuable tool for the physician to be guided to prescript efficient antiretrovirals and not to prescribe a drug that is not efficient and is expensive, toxic and with secondary effects for the seropositive patient


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